The recent advances in drug discovery and molecular and pharmaceutical biology have created a need for the development of effective mechanisms for delivering therapeutic agents into cells. In but one example, researchers have particularly struggled to develop efficient means for introducing nucleic acids into cells. The development of a method to efficiently introduce nucleic acids into cells would be useful, for example, in gene therapy, antisense therapy, research purposes (e.g., to study cell differentiation, growth and carcinogenic transformation or for the creation of animal models for human disease; see, for example, Abdallah, Biol. Cell, 1995, 85, 1, and references therein).
An efficient way in which nature has accomplished the task of delivering biological agents into cells is through the evolution of viruses specifically to mediate the transfer of genetic materials into cells. Although viruses are ideal vectors for gene therapy because they have the highest levels of transfection efficiency known, the human immune system has likewise evolved to counteract viral infections, thus making virus-based gene therapy in humans potentially unsafe. Because of this characteristic of viral gene delivery systems, non-viral, or synthetic, gene delivery systems have been created to mediate the transfer of therapeutic agents into cells. Unfortunately, existing techniques for delivering nucleic acids to cells are limited by poor efficiency and/or high toxicity of the delivery reagents. A particular problem is encountered with techniques that rely on receptor-mediated endocytosis because the nucleic acid to be delivered is often destroyed when exposed to the low pH and active degradatory machinery of the endosome/lysosome. Various reagents (e.g., chloroquine, polyethyleneimine [PEI], certain highly charged cationic compounds, fusogenic peptides, and inactivated adenoviruses) have been developed that are intended to quickly disrupt the endosome in order to minimize the amount of time that a delivered nucleic acid spends in this hostile environment. Specifically, two delivery systems that have obtained recognition as the forerunners for in vivo gene therapy are those based on polymers and on lipids.
The first polymer used for a gene delivery system was polylysine (Wu, G. Y., Wu, C. H. J Biol. Chem. 1988, 263, 14621), in which the polylysine was complexed with plasmid DNA and evaluated for its ability to mediate the transfection of cells. Polylysine continues to be a common choice for the study of gene transfer, however, the gene expression mediated by polylysine, and other like polycations, is low. In an effort to increase the transfection efficiency of polylysine and other like polycations, agents designed to destabilize the endosome have been typically used in conjunction with lysine. One example of such an agent is chloroquine, which is a weak base that buffers the endosome to maintain a neutral endosomal pH. This buffering effect serves two functions to increase transfection efficiency (Seglen, P. O. “Inhibitors of lysosomal function” Methods Enzymol. 1983, 96, 737). First, the enzymes of the lysosome (which fuses with the endosome to form a secondary lysosome) have maximum activity in an acidic medium. Therefore, buffering of the vesicle diminishes the activity of the enzymes including nucleases that can degrade the therapeutic plasmid DNA. Second, the buffering effect may also act to cause vesicle expansion and subsequent destabilization of the endosome. While chloroquine provides an effective system to study the transfection efficiencies of delivery systems in vitro, its use in vivo is unrealistic owing to the high local concentrations required to increase the gene expression. One substitute utilized for chloroquine includes glycerol, which has recently been shown to enhance gene expression in vitro. However, 1-1.5 M glycerol is required to enhance transfection, thus also making glycerol impractical for in vivo gene transfer Zauner, W.; Kichler, A., Schmidt. W.; Sinski, A.; Wagner, E. Biotechniques 1996, 20, 905). Other approaches used to increase the gene expression by polylysine/DNA conjugates rely on virus or virus components. For example, the addition of adenovirus to a polylysine/DNA complex increased transfection levels over 2000 times in vitro (Wu, G. Y.; Zhan, P.; Sze, L. L.; Rosenberg, A. R.; Wu, C. H. J. Biol. Chem. 1994, 269, 11542), and conjugation of endosomolytic peptides to polylysine/DNA complexes increased transfection efficiency more than 1000 times in vitro (Plank, C.; Oberhauser, B.; Mechtler, K.; Koch, C.; Wagner, E. J. Biol. Chem. 1994, 269, 12918). However, the inclusion of potentially immunogenic protein-based endosomolytic agents into gene delivery systems may significantly diminish the utility of the conjugate for repeated use in vivo.
Another synthetic polymer that has been recently evaluated for its ability to mediate gene transfer is polyethyleneimine (PEI) which contains primary, secondary, and tertiary amines (Abdallah, B.; Hassan, A.; Benoist; Goula, D.; Behr, J. P.; Demeneix, B. A. Human Gene Therapy 1996, 7, 1947; Boussif, O.; Lezoualc'h, F.; Zanta, M. A.; Mergny, M. D., Scherman, D.; Demeneix, B.; Behr, J. P. Proc. Natl. Acad. Sci. USA 1995, 92, 7297). The advantage of PEI over polylysine as a mediator for gene transfer is that it contains a built-in mechanism for endosome escape. The overall protonation of PEI at pH=7 is approximately 20% whereas the overall protonation at pH=5 is approximately 45% (Suh, J.; Paik, H. J.; Hwang, B. K. Bioorg. Chem. 1994, 22, 318). These different protonation levels suggest that PEI is capable of buffering the endosome in the same manner as chloroquine. In fact, PEI is capable of mediating gene transfer equal to the best lipid systems. However, while PEI demonstrates that the synthetic “built-in” mechanism for endosomal rupture works well to mediate gene transfer, PEI is toxic to cells in vitro (Personal experimental evaluation, MIT, April 1998).
In addition to synthetic polymers, lipids have also been extensively studied for uses in gene therapy. These lipids are generally cationic lipids containing a positively charged head group and a hydrophobic tail. The first widely recognized lipid-based DNA delivery system was a mixture of DOTMA (N-1,2,3-dioleyloxy)propyl]-N,N, N-trimethylammonium chloride) and DOPE (dioleoyloxy phosphatidylethanolamine) known as Lipofectin (Felgner, P. L.; Gadek, T. R.; Holm, M.; Roman, R.; Chan, H. W.; Wenz, M.; Norhrop, J. P.; Ringold, G. M.; Daneilsen, M. Proc. Natl. Acad. Sci. USA, 1987, 84, 7413). Since Lipofectin was introduced many lipid-based gene transfer systems were synthesized, such as DOGS (dioctadecylamidoglycylspermine), DDAB (dimethyldioctadecylammonium bromide) and DOTAP ([1,2-dioleoyl-3-trimethylammonium-propane). Although lipid-based systems generally have transfection efficiencies superior to polymer-based systems, their levels of transfection are still generally insufficient for practical clinical somatic gene therapy.
Although attempts have been made to improve each of these known endosomolytic agents, they still possess toxicity problems and other disadvantages. Clearly, a versatile and biocompatible synthetic cell delivery system, preferably biodegradable, would be important for the clinical success of gene delivery and the delivery of other therapeutic agents.